This invention is related to host marking pheromones (HMPs) also known as oviposition deterring pheromones (ODPs) in insects. In particular it refers to host marking pheromones in fruit flies of the genus Anastrepha.
Fruit flies (Diptera: Tephritidae) are considered among the most economically important pests worldwide (Aluja and Liedo 1993; McPheron and Steck 1996). The most notorious pestiferous species belong to the genera Anastrepha, Bactrocera, Ceratitis, Rhagoletis, and Toxotrypana (Aluja 1993). Among the 184 reported species of Anastrepha (Aluja 1994), seven stand out because of the damage they cause to commercially grown fruit: A. fraterculus, A. grandis, A. ludens, A. obliqua, A. serpentina, A. striata and A. suspensa. Distribution and fruit species attacked are indicated in TABLE 1 (from Hernandez-Ortiz and Aluja 1993).
Damage of these fly species is direct (larvae in fruit) and indirect (severe quarantine restrictions that limit international commerce). Infestation level (i.e., percent infested [=lost] fruit in a tree can vary between zero and 90% depending on the fruit growing region, fruit species or cultivar, size of fruit fly population, management intensity in orchard and degree of capitalization of orchard owner. Control of these pests has been historically attempted through fumigants, toxic bait sprays (a food-based bait mixed with-an insecticide) and on occasion by use of the sterile insect technique (SIT) (reviewed by Steiner 1955 and Aluja 1994). Despite being quite effective, the large scale use of toxic bait sprays is no longer acceptable because of the negative impact on beneficial and native entomofauna (Asquith and Messing 1992, Hxc3x6lmer and Dahlsten 1993 and references therein). In recent years, a number of alternatives such as the use of gibberelic acid to enhance the innate resistance of citrus to fly attack (Greany 1989), insect growth regulators such as cyromazine (Moreno et al. 1994), pathogens such as Bacillus thuringiensis Berliner (Martinez et al. 1997) and photoactivated dyes as for example SureDyeM.R. (PhotoDye International, Inc., Boca Raton, USA) are being explored. Despite their promise as viable alternatives to toxic bait sprays, some of these methods could still prove unacceptable because of the deleterious effect on nontarget insects (Aluja 1996). The latter, because the killing agent needs to be ingested by the adult insect. The only practical way of achieving this is by mixing it with a food-based bait. As is the case with food-based baits mixed with an insecticide, lures used in combination with photoactivated dyes or insect growth regulators are non-specific. That is, they attract a large, number of nontarget insects (such as many species of insects in the order Diptera) which are also killed.
A highly selective alternative to the use of insecticides that has been recently tested in fruit flies of the genus Rhagoletis, and that does not require a bait to be effective, is the use of synthetic host marking pheromones (HMPs). HMPs are deposited by flies on the surf ace of a fruit after an egglaying bout and given a large enough concentration deter conspecifics from ovipositing in the same fruit (Katsoyannos and Boller 1980). Based on this knowledge and chemical work by Hurter et al. (1987a; 1987b) and Ernst and Wagner (1989), it was possible to successfully test the synthetic HMP of Rhagoletis cerasi as a fruit-infestation-reducing-agent in commercial cherry orchards in Switzerland (Aluja and Boller 1992a; 1992b). Application of synthetic HMP to the entire tree crown reduced the number of larvae per kg of fruit by a factor of 10 when compared with an untreated tree (0.226 vs. 0.021 pupae/fruit) in untreated and treated trees, respectively. A significant reduction.in fruit infestation could also be achieved when only one half (top to bottom) of the tree crown was treated (Aluja and Boller 1992b). Of significance here, is the fact that host marking behavior has been also reported in several Anastrepha species: A. suspensa (Prokopy et al., 1977), A. fraterculus (Prokopy et al. 1982), A. sororcula and A. obliqua (Simoes et al. 1978), A. pseudoparallela (Polloni and Da Silva 1986), A. striata (Aluja et al. 1993), A. bistrigata (Gomes -Da Silva 1991), A. grandis (Selivon 1991) and A. ludens (Papaj and Aluja 1993). As was the case with Hurter and collaborators (Hurter et al. 1987b), when working with R. cerasi, Santiago and collaborators (1990; 1991) demonstrated that the feces of A. ludens and A. serpentina contained a HMP. Using thin-layer chromatography, they further showed that one band provoked the deterrent effect. They also were able to show that crude feces extracts of A. ludens applied to fruit-bearing mango tree branches, reduced the level of infestation by A. ludens (Santiago et al. 1991). In general, host marking pheromones, given a high enough concentration, deter oviposition in fruit by fruit fly females (Averill and Prokopy 1989).
There remains a need in the art for a highly selective (i.e., directed only at flies in the genus Anastrepha) and environmentally-friendly alternative to the use of Anastrepha control methods that is not dependent on a food-based bait to deliver the toxicant or killing agent. The present invention describes several substances that reduce the damage inflicted to fruit of.value to humans by flies in the genus Anastrepha and that does not require a food-based bait to be delivered or to be effective.
According to the present invention it has been found that 2-(2xe2x80x2, 14xe2x80x2-Dimethyl-pentadecanoylamino)-pentanedioic acid of the formula (I), 
isolated from the feces of Anastrepha ludens, functions as an oviposition deterrent against economically and non-economically important fruit flies (Diptera: Tephritidae) of the genus Anastrepha. This is significant because the above mentioned substance if properly formulated, can henceforth be used to reduce the damage these insects inflict on fruit grown in commercial and semi-commercial orchards, in backyard gardens or in single trees planted in residential gardens. The present invention relates to a method for the isolation of the host marking pheromone (oviposition deterring pheromone) of Anastrepha ludens which is applicable to all species of fruit flies of the genus Anastrepha. The invention also relates to a method for the synthesis of oviposition deterrents which have the general formula (II) 
Where R1 is H, C1-C4 alkyl, C3-C6 cycloalkyl, C3- or C4 alkenyl, C3- or C4 alkynyl;
R2 and R3 independent of one another are H or C1-C4 alkyl, C3-C6 cycloalkyl, C3- or C4 alkenyl, C3- or C4 alkynyl, benzyl or benzyl which is substituted once to three times on the phenyl ring by halogen, C1-C4 alkyl;
R4 is H or C1-C4 alkyl, C3-C6 cycloalkyl, C3- or C4 alkenyl, C3- or C4 alkynyl, C1-C4 alkyl carbonyl, benzyl or benzyl which is substituted once to three times on the phenyl ring by halogen, C1-C4 alkyl,
Where xcex1 refers to (R) or (S) stereoisomers (or their mixtures), with the premise that R1 is not=H.
Where n=an integer between 0 and 15.
Where * refers to (L) or (D) amino acid stereochemistry (or their mixtures).
The alkyl, alkenyl and alkynyl groups in the above definitions can be straight chain or branched. Alkyl groups for example are methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, and tert-butyl.
Examples of alkenyls are vinyl, allyl, methallyl, 1-methylvinyl, but-2-en-1-yl.
The cycloalkyl radicals which are suitable substituents are, for example cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
Alkyl carbonyl is, for example acetyl, propionyl and pivaloyl.
The invention also relates to the mono- or di-carboxylic acid salts which the compound of formula (II) can form with bases. These salts are for example, alkali metal salts, for example sodium and potassium salts; alkaline earth metal salts, for example calcium and magnesium salts; ammonium salts, i.e. unsubstituted ammonium salts and mono or polysubstituted ammonium salts, for example triethylammonium and methylammonium salts; or salts with other bases.
Preferred alkali metal and alkaline earth metal hydroxides as salt-forming agents are, for example, the hydroxides of lithium sodium, potassium, magnesium or calcium, and in particular those of sodium and potassium.
Examples of amines which are suitable for ammonium salt formation include ammonia and primary, secondary, and tertiary C1-C18 alkyl amines, for example methylamine, ethylamine, n-propylamine, diethylamine, and triethylamine; Preferred is triethylamine.
The possible presence of at least one asymmetric carbon in the compounds of formula (II) means that the compounds can occur in optically active individual isomers and in the form of racemic mixtures. In the present invention, the active compounds of the formula (II) are to be understood as meaning both the pure optical antipodes and the racemates or diastereoisomers.
If an aliphatic Cxe2x95x90C double bond is present, geometric isomerism can occur. The present invention also relates to these isomers.
Preferred compounds of the formula (II) have the formula (IIa) 
in which R1, R2, R3, n, xcex1 and * are as defined under formula (II).
Particularly preferred compounds are those of the formula (IIa) in which R1 is methyl, and n is an integer between 5 and 10.
Compounds which are also especially preferred are those of the formula (IIa) where R2 and R3, independent of one another, are H or methyl.
Compounds which are also especially preferred have (R) or (S) xcex1 stereochemistry or are mixtures of xcex1-(R)/(S) isomers.
Compounds which are particularly important have (L) stereochemistry at *.
The raw material was extracted from fly feces of laboratory reared A. ludens, A. obliqua and A. serpentina. Feces were obtained as follows: In a 30xc3x9730xc3x9730 cm glass cage, we placed 300 ml of fruit fly pupae (equivalent to ca. 11,000 adult flies). In order to increase the exposed surface to flies, two 13xc3x9725 cm glass pieces were introduced into the cage. Each cage was provided with food (sucrose and hydrolyzed protein at a 3:1 ratio) and water. Flies were kept in cages during 30 days. At this time, all living and dead flies were removed. Dried eggs, broken wings, and legs were also removed. The remaining xe2x80x9cdirtxe2x80x9d (containing mostly fly feces) was then scratched off the glass surfaces with a metal spatula. Each cage yielded ca. 10 g of feces. Fly feces were kept in plastic petri dishes or bottles at xe2x88x9215xc2x0 C. until further use. To obtain crude pheromonal extracts for purification and biological experimentation batches of 400 g of feces of Anastrepha ludens were mixed with 1,000 ml of methanol and sonicated manually for 15 min. After this, the liquid was subjected to centrifugation at 12,000 RPM during 20 min. The supernatant was then concentrated in a rotary evaporator to provide the stock solution for further use.
Extraction and purification procedure of the oviposition deterring, host marking pheromone
167 g fly feces from Anastrepha ludens were suspended in 5 l of ethanol and stirred for 17 hr. at room temperature. The solid material was filtered off, rinsed with 1 l ethanol and the extraction procedure was then repeated once with 2 l ethanol containing 3.5 ml trifluoro-acetic acid. The combined ethanol extracts were concentrated on a rotary evaporator at 50xc2x0 C. and 20 mbar to almost dryness. After 6 hr. of lyophilization, the residue (33.8 g) was dissolved in 300 ml methanol at 50xc2x0 C. and cooled down to room temperature. After 2 hr., the precipitated fat (10.5 g) was filtered off and rinsed with methanol. The solution was then evaporated to dryness, giving 23.3 g of a honey-yellow residue which was used in four batches for preparative HPLC.
Column 1: 50xc3x97250 mm, Lichrospher RP-18, 7 xcexcm (Merck). Flow rate: 0-60 min.: 75 ml/min., 60-90 min.: 100 ml/min. Mobile phase: 0-60 min., linear gradient from 100% water to 100% methanol, 60-90 min.: 100% methanol. The fractions were collected according to peak development of chromatogram. UV-Detection: 220 nm. Electrophysiological activity eluted between 47-60 min. containing 2.43 g of dry matter.
Column 2: 50xc3x97250 mm, Kromasil KR100-C18, spher. 7 xcexcm (Eka Nobel), Flow rate 0-45 min.: 70 ml/min., 45-90 min.: 100 ml/min. Mobile phase: 0-45 min.: linear gradient from 50% acetonitrile in water to 100% acetonitrile. Fractions were collected according to peak development of chromatogram. UV-Detection: 200 nm. Two electrophysiological active regions: (1) 12-18 min., containing 63 mg dry matter, and (2) 36-42 min., containing 28 mg dry matter.
For further investigations, only material from region 1 was purified.
Mobile phases consisted of the following solutions:
A: 100% of water with 0.1% of formic acid.
B: 100% of acetonitrile with 0.1% of formic acid.
Detection: UV at 195 nm.
HPLC columns were supplied by: Macherey-Nagel.
Column 3: 10xc3x97250 m, 10 xcexcm Nucleosil CN 100. Flow rate: 4 ml/min. Injection: 6.3 mg in 1.0 ml of water (10 repetitions). Mobile phase: 0-2 min., 80% A in 20% B; 2-25 min., linear gradient from 80% A in 20% B to 60% A in 40% B; 25-35 min 60% A in 40% B; 35-40 min., linear gradient from 60% A in 40% B to 100% B. Vol. of fractions: 4 ml. Electrophysiological activity in fractions 23-27, evaporated (Rotavap), dissolved in 1 ml of 50% A in 50% B.
Column 4: 10xc3x97250 mm, 7 xcexcm Nucleosil Phenyl 100. Flow rate: 4 ml/min. Injection 200 xcexcl (5 repetitions). Mobile phase: 0-60 min., 68% A in 32% B. Vol. of fraction: 4 ml. El. act. in fractions 36-42, evap. and dissolved in 5 ml of 50% A in 50% B.
Column 5: 10xc3x97250 mm, 7 xcexcm Nucleosil C-18 100. Flow rate: 4 ml/min. Injection 1 ml (5 repetitions). Mob. phase: 0-60 min. 65% A in 35% B. Vol. of fractions: 4 ml. El. act. in fractions 34-40, evap. and dissolved in 1.0 ml of 50% A in 50% B.
Column 6: 4xc3x97250 mm, 7 xcexcm Nucleosil OH (Diol) 100. Flow rate: 1.0 ml/min. Injection: 200 xcexcl (5 repetitions). Mobile phase: 0-70 min., 65% A-in 35% B. Vol. of fraction: 1.0 ml. El. phys. act. in fractions 9-11, evap. and dissolved in 1.0 ml of 50% A in 50% B.
Column 7: 4xc3x97250 mm, 5 xcexcm Nucleosil C-18 AB 100. Flow rate: 1.0 ml/min. Injection: 250 xcexcl (4 repetitions). Mobile phase: 0-70 min., 65% A in 35% B. Vol. of fraction: 1.0 ml. El. phys. act. fractions 49-52 combined and evap. to dryness for structural analysis.
Structure elucidation of the oviposition deterring, host marking pheromone
Mass Spectroscopy
FAB-MS gives strong molecular ions at m/z 400 (MH+) and 422 (M Na+) this corresponds to a molecular weight of 399 for the pheromone. The exact mass has been determined by high resolution MS to 422.2864 for M+Na, and the molecular formula could be deduced to C22H41NO5 for the pheromone with a difference of 1-8 uma from calculated to observed mass.
The pheromone has been esterified with diazomethane giving a molecular ion at m/z 428 (MH+, APCI-MS). The difference of 28 mass units indicates the presence of two methylated carboxyl groups. Characteristic fragmentations are observed for the molecular ion in the MS-MS mode. All of the recorded fragments can be assigned to specific cleavages (relative intensities in % of the base peak). 428(77), 396(31), 368(8), 336(1), 253(6), 225(5) 183(2), 176(100), 169(6), 158(45) 155(4), 144(42), 141(4), 127(6), 116(19), 113(4), 99(4), 98(17), 85(3), 71(4), 57(4), 43(1).
Chemical shifts of the isolated natural pheromone in ppm, 500 MHz, in CDCl3:
The NMR signals in methanolic solvents are markedly sharper. The chemical shifts of the natural pheromone in CDCl3/CD3OD 1:1 are:
1H-NMR: 7.60 (s, 1 NH), 4.36 (dd, 1H), 2.32 (m, 2H), 2.27 (m, 1H), 2.11 (m, 1H), 1.88 (m, 1H), 1.52 (m, 1H), 1.42 (m, 1H), 1.29 (m, 1H), 1.18 (br., 18H), 1.07 (q, 2H), 1.03 (d, 3H), 0.77 (d, 6H).
13C-NMR(CDCl3=77.0 ppm): 177.99, 175.04, 173.5, 51.18, 40.45, 38.49, 33.56, 29.76, 29.29, 29.05, 29.00 (4xc3x97), 28.89, 27.34, 26.84, 26.76, 26.32, 21.71 (2xc3x97), 16.67.
Proton and carbon connectivities are based on two-dimensional NMR experiments (COSY, HCCORR, HMBC, ROESY,).
Chirality Determination of the Amino Acid Moiety
The acid hydrolyzed natural pheromone has been derivated to the N-trifluoroacetyl glutamic acid isopropylester. On the chiral GC column (Chirasil-L-Val), the L-isomer showed a retention time of 25.6 min., the D-isomer 24.2 min. based on the synthetic compounds. Temperature program: 70xc2x0 C. (3 min. isocratic), 2xc2x0 C./min. to 190xc2x0. The sample of the chromatographically purified natural product consisted of 21% D-glutamic acid and 79% L-glutamic acid which has been confirmed by co-injection of the natural and synthetic sample.
Chirality Determination of the xcex1-methyl Fatty Acid Moiety
The four possible stereoisomers can be described as R-L, S-L, R-D, S-D, in which R and S describe the chirality of the xcex1-methyl fatty acid, whereas L and D indicate the chirality of the glutamic acid.
The total synthesis of all four possible isomers has been achieved via stereoselective routes analogous-to literature procedures as described below. The two diastereomeric pairs of enantiomers can be distinguished under specific HPLC conditions (Nucleosil-100-7um-Phenyl, 10xc3x97250 mm, 4 ml/min, 40% acetonitrile/60% water, 0.1% formic acid, 195 nm UV detection). The retention time for R-L and S-D was 46 min. whereas R-D and S-L had 48 min. The natural pheromone eluted after 46 min. Since the amino acid configuration of the major component of the natural product was determined as L, the chirality of the a-methyl group of the isopalmitic acid part was unambiguously assigned as R. This has been confirmed by co-injection of the natural pheromone and the synthesized compound.
Formula I. Chemical structure of the natural host marking pheromone (HMP) of Anastrepha ludens indicating carbon numbering. 
Where the stereochemistry at C-2 is 79:21 (L):(D)
The process according to the invention for the preparation of compounds of the formula (II) is carried out analogously to known literature procedures and involves either
a) reaction of a compound of the formula (III) 
xe2x80x83where R1, R2, R3, n and xcex1 are as defined under formula (II), where X is an acid activating group, for example halogen, in an inert organic solvent in the presence of a base, with a compound of the formula (IV) 
xe2x80x83in which R4 and * are as defined in formula (II), and where R5 represent cleavable protecting groups, to give the compounds of the formula (V) 
xe2x80x83in which the protecting groups R5 of these compounds are cleaved and replaced by H, or
b) reaction of a compound of the formula (III) 
xe2x80x83where R1, R2, R3, n and xcex1 are as defined under formula (II), and X is an acid activating group, for example halogen, in an inert organic solvent in the presence of a base, and a solubilizing agent, for example lithium chloride, with a compound of the formula (VI) 
xe2x80x83in which R4 and * are as previously defined.
Compounds of the formula (VI) are glutamic acid derivatives and are widely available. Compounds of the formula (IV) contain protecting groups R5 which are typical protecting groups used in peptide, chemistry. Examples of such are C1-C4 alkyl; benzyl which is substituted once to three times on the phenyl ring by halogen, C1-C4 alkyl; C3-C4 alkenyl.
The alkyl, alkenyl and alkynyl groups in the above definitions can be straight chain or branched. Alkyl groups for example are methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, and tert-butyl. As an example of an alkenyl protecting group, allyl is preferred.
Especially preferred are compounds where R5 is benzyl.
The protecting groups can be introduced and removed by known methods (see for example Greene, 1981). For example a compound of the formula (V) where R5 is benzyl, deprotection can be achieved by standard catalytic hydrogenation, or by catalytic hydrogen transfer hydrogenation as described in the literature (Means et al., 1979).
The activating groups X used for the coupling of compound (III) with compounds (IV) and (VI) are, for example halogen, or activated esters well known in peptide synthesis (see for example Geiger, 1985). Preferred methods for activation of the acid (compound (III), Xxe2x95x90OH) are formation of the acid chloride, using for example thionyl chloride with a catalytic amount of dimethylformamide, or formation of the activated esters using, for example, N-ethyl-Nxe2x80x2-(3-dimethylaminopropyl)-carbodimide (EDC) or dicyclohexylcarbodiimide. The reaction of compound (III) with compound (IV) is carried out in an inert organic solvent, for example, a chlorinated hydrocarbon solvent such as dichloromethane, or an aromatic hydrocarbon solvent, such as toluene, in the presence of a base, for example an alkyl amine such as triethylamine or an aromatic amine, for example 4-dimethylaminopyridine (DMAP), or a combination of bases. The reaction can be carried out at temperatures between 0 and 120xc2x0 C. Reaction of compounds of the formula (VI) with compounds of the formula (III) are carried out in the presence of a solubilizing agent such as LiY, where Y is a halogen, for example chloride, in an inert ether solvent, such as tetrahydrofuran, at temperatures between 0 and 120xc2x0 C. The use of solubilizing agents in peptide chemistry is well known in the literature (Seebach et al., 1995).
Compounds of the formula (III) containing an xcex1 substituent group can be prepared in racemic form according to literature methods (see for example Hoefle et al., U.S. Pat. No. 4,716,175 A, 1987). The compounds of formula (V) are subsequently formed as mixtures of diastereoisomers and may be separated by, for example HPLC methodology.
Alternatively compounds of formula (III) (where R1 is not H) may be prepared in a stereospecific manner by attaching a group containing at least one chiral center (a chiral auxiliary) to a compound of formula (III) (where R1 is H), which leads to a chiral compound of formula (VII), which can then be reacted with a compound R1-Y, where R1 is as previously defined, with the premise that it is not H, and Y is halogen, to give a compound of formula (VIII), and then removal of the chiral auxiliary, to give compounds of the formula (III). Subsequent activation and reaction of these compounds with compounds of the formula (IV) or (VI) leads to compounds of the formula (II) or (V), where R1 is not H, in non-racemic form, as illustrated in Scheme 1. 
The use of chiral auxiliaries in stereospecific synthesis is well known (Ager et al., 1995). Chiral auxiliaries are, for example, 2-(R)- or 2-(S)-Bornane-10,2-sultams, 
which can be coupled to compounds of formula (IIIa), where X is an activating group for example chlorine, and where R2, R3 and n are as defined in formula (II), and R1 is H, to give compounds of the formula (VIIa) and (VIIb). 
Compounds of formula (VIIa) can be deprotonated with a strong base, for example n-butyl lithium, in an inert solvent, such as tetrahydrofuran, and a dipolar aprotic co-solvent, such as 1,3-dimethyl-tetrahydro-2-(1H)-pyrimidone (DMPU), and subsequently reacted with.an alkylating agent R1-Y, where R1 is as previously defined, with the premise that it is not H, and Y is halogen, for example iodide, to give a compound (VIIIa), preferentially of the R1-xcex1-configuration illustrated for (VIIIa). 
Analogously, compounds of formula (VIIb) can be reacted with compounds R1-Y to give compounds of type (VIIIb), where the R1-xcex1-configuration is predominantly as shown in (VIIIb). 
Face selective alkylations of this type are predictable and well documented in the literature (Oppolzer et al., 1989).
The chiral auxiliaries can be removed from compounds of the formula (VIII) by, for example, hydrolysis by a base, for example lithium hydroxide, in an inert solvent, such as tetrahydrofuran, and an a protic solvent, such as water, in the presence of a inorganic peroxide, such as hydrogen peroxide, analogously to literature procedures (Evans et al., 1987). After activation, compounds of formula (III), where X is an activating group, for example chlorine, can be used to prepare compounds of the formula (II) with defined xcex1-stereochemistry, based upon the mechanistic discussions of Opollzer et al., (1989).
One skilled in the art will realize that compounds of the formula (II) can be prepared according to this process with defined stereochemistry at the xcex1 and * positions by judicious choice of the chiral auxiliary, and the L- or D-isomers of compounds of formula (IV) or (VI).